We previously constructed a first-generation construct called HPIV3-EbovZ GP, in which the complete genome of the JS strain of HPIV3 was modified by the addition of the EBOV GP gene in the third gene position, between the HPIV3 P and M genes. The JS strain is thought to be an attenuated HPIV3, based on previous clinical studies, although the basis of this attenuation is unknown. EBOV GP is the sole EBOV virion surface protein, the sole EBOV neutralization antigen, and the major protective antigen. The EBOV GP gene was engineered to have the appropriate HPIV3 transcription signals for it to be expressed as a separate mRNA by the HPIV3 polymerase. HPIV3-EbovZ GP was substantially immunogenic and protective when given to non-human primates by combined intranasal (IN) and intratracheal (IT) administration, even in animals previously infected with HPIV3. However, immunogenicity depended on IT delivery of vaccine: IN delivery alone was insufficient. This suggested that vector expression beyond the upper respiratory tract was necessary for immunogenicity. We therefore explored delivery of the HPIV3-EbovZ GP construct by the aerosol route in rhesus macaques. The aerosol route was generally more immunogenic and protective than the combined IN/IT route. This induced generally higher serum and mucosal EBOV-specific IgG, IgA, and neutralizing antibody titers, as well as EBOV-specific cellular responses in the lungs, including polyfunctional CD8+ T cells and CD4+ T helper cells that were predominately Th1. In addition, the HPIV3-EbovZ GP vaccine induced more robust cell-mediated and humoral immune responses than an alphavirus vaccine delivered parenterally in parallel. One aerosol dose of HPIV3-EbovZ GP conferred 100% protection to macaques against EBOV challenge. We developed a second-generation version of this vector, called HPIV3/delHNF/EbovZ-GP, in which the HPIV3 F and HN genes were deleted, leaving EBOV GP as the sole viral surface glycoprotein. A large comparative study in cynomolgus monkeys by our collaborator Alexander Bukreyev at the University of Texas Medical Branch, Galveston, (who made the construct while a Staff Scientist in LID/NIAID) showed that this second-generation version was even more protective than the first-generation even though it was very highly restricted for replication (much more restricted than the first-generation construct). Nine different pneumovirus or paramyxovirus respiratory viral vectors expressing EBOV GP were compared for immunogenicity and protective efficacy in a guinea pig model for EBOV infection. These vectors included the two HPIV3-based constructs described above (HPIV3-EbovZ-GP and HPIV3/delHNF/EbovZ-GP), two comparable HPIV1-based constructs, and five NDV-based constructs. This study was done primarily by our collaborators at the University of Texas Medical Branch, Galveston. They comprehensively evaluated the antibody responses to the panel of nine respiratory EBOV vaccines given intranasally. Eight of the vaccines were completely protective in guinea pigs, but the vaccines yielded antibody repertoires that differed in a number of their properties, including: avidity towards GP and its fusogenic form, targeting of key antigenic regions, neutralizing antibody specificities, and linear epitope preferences. Competition studies with monoclonal antibodies from human survivors demonstrated that the magnitude of antibodies recognizing the receptor-binding domain and the GP1/GP2 interface at the base of GP correlated with neutralizing titers. These unexpected differences showed that, while an immunogen may determine the general target of an antibody response, distinct vaccine vectors can induce quantitatively and qualitatively different responses that can affect protective efficacy. These data suggest that immune correlates of vaccine protection cannot be generalized for all vaccines against the same pathogen, even if they use the exact same immunogen. We performed (with clinical collaborators at the Johns Hopkins Bloomberg School of Public Health) an open label phase 1 clinical trial to determine the safety, tolerability, and immunogenicity of HPIV3-EbovZ GP delivered IN in healthy adults in an inpatient setting (NCT025645750), which was intended to be a safety study prior to evaluating aerosol delivery. Ten subjects received two doses (4- to 8-week interval) of 6.0 log10 PFU of vaccine. The first dose was moderately infectious (7/10 subjects shed virus detected by qRT-PCR, mean peak titer 3.8 log10 genomic equivalents/ml, mean duration of shedding 7.9 days). Little shedding was detected after the second dose. A second cohort (n=20) received one of two planned doses of 7.0 log10 PFU of vaccine. Shedding was similar but of shorter duration (mean of 3.7 days). The vaccine was well tolerated, with the exception that asymptomatic ALT elevations were noted in 5 volunteers (3 mild, 2 moderate) in cohort 2 after vaccination and associated with shedding. All resolved by day 28. The study was halted due to these elevations of ALTs, but their significance is unclear. Because of this, this vaccine will not be administered further at this time. Induction of serum antibodies was poor (mucosal antibodies not yet analyzed), but this was expected since, as noted above, we had previously observed that administration by the IN route alone was poorly immunogenic in rhesus monkeys. We have initiated a Phase 1 study to evaluate the safety, infectivity, and immunogenicity of two doses of the HPIV3/HNF/EbovZ GP vaccine candidate when administered intranasally in healthy adults in an inpatient setting (NCT03462004). Participants are being enrolled sequentially in two cohorts. Participants in Cohort 1 have been randomly assigned to receive two doses of either 6.0 log10 PFU/mL of HPIV3/delHNF/EbovZ-GP vaccine or placebo. The first dose was given on Day 0 and the second dose was given 35 days later. Vaccine replication was evaluated by nasal wash and RT-qPCR and infectivity assays, and serum antibody responses will be measured. As expected, at the 6.0 log10 PFU dose, the HPIV3/HNF/EbovZ-GP vaccine was marginally infectious, and adverse events were generally mild to moderate. The study was deemed safe to proceed to the evaluation of the higher 7.0 log10 PFU dose. Participants in Cohort 2 will be randomly assigned to receive two doses of either 7.0 logPFU/mL of HPIV3/HNF/EbovZ-GP vaccine or placebo on Days 0 and 28.